Methods: :
Three independent cultures of PTM cells were challenged with1 x 107 particles of different phagocytic ligands (FITC-labeledE.coli and fluorescent latex beads) for 2, 5, and 10 days. Phagocyticcapacity was evaluated by fluorescence microscopy. Lysosomalcontent was quantified by flow cytometry using lysotracker red.Co-localization of phagocytic ligands within the lysosomal compartmentwas monitored by confocal microscopy. Ultrastructural analysiswas performed by transmission electron microscopy. Kineticsof phagosome maturation as a function of phagosome/lysosomefusion was monitored using E.coli labeled with the pH sensitivedye pHrodo. Cathepsin levels were quantified by qPCR and WBanalysis. Cathepsin activities were evaluated using fluorogenicsubstrates (z-FR-AMC, z-RR-AMC, z-GPR-AMC, z-VVR-AMC, CTSD/Esubstrate).

Results: :
PTM cells showed an innate capacity to ingest large amountsof inert and biologically active particles, without significantlyaffecting cell viability; only slight cytotoxicity was observedwith E. coli at Day 10. Electron micrographs revealed the presenceof multivesicular bodies, phagolysosomes, as well as intracellularand extracellular membranous figures following phagocytic challenge.Co-localization of ingested particles within lysosomes and increasedlysosomal content (40.17% ± 5.67, p<0.05, n=3) wereobserved with E. coli, but not with beads. Kinetics analysisshowed that E. coli reached the lysosomal compartment withinthe first 2 hours. Phagocytosis of E. coli was significantlyreduced in the absence of serum, suggesting Fc receptor-mediatedphagocytosis. The mRNA and activity levels of several cathepsins(K, L, D, and B) and genes related with the autophagy/lysosomalpathway (ATG6, LAMP-2) were upregulated with phagocytic stress.The most striking changes were the sustained dramatic increaseof cathepsin B expression (mRNA and protein levels; 2.64±0.5fold, 5.03±1.46 fold, and 8.76±2.55 fold at day2, 5, and 10, respectively; p<0.05, n=3) and cathepsin Bactivity (77.2±12.1%, 82.4± 7.6%, and 94.1±11.3%at day 2, 5, and 10, respectively; p<0.05, n=3) in E. colichallenged-cultures.

Conclusions: :
Our results indicate that although PTM cells are able to efficientlyphagocytose different materials, the fate and elicited responsesvary among the nature of the ingested particles. A better understandingof the phagocytic/lysosomal functions in TM cells is particularlyimportant given the potentially critical role of ingestion/degradationof extracellular material in outflow pathway function.